Abstract
Histone deacetylases (HDACs) are essential drug targets, making HDAC inhibitors (HDACi) valuable therapeutics for cancer and cardiovascular diseases. Most inhibitors used are pan-inhibitors including FDA-approved or those that are in clinical trials. This non-specific targeting often leads to off-target effects and cytotoxicity. Hence, it is crucial to develop a high-throughput method for screening HDACi) to determine specificity, identify off-target effects, and target HDAC activity to a specific gene, thereby developing targeted therapy. Herein we combined both proteomics and genomics approaches for mass spectrometry screening of HDACi to identify histone modification changes and understand the transcriptional regulation of H2A.Z acetylation in breast cancer cells. Upon screening about 50 HDACi including SAHA, Panobinostat, Romidepsin, Entinostat, TSA, SB939 and Belinostat, a global increase in histone acetylation was observed. We observed Entinostat-induced histone H2A.Zac at positions K4, K7 and K11 in both HeLa and breast cancer cell lines (MCF7, MDA-MB-231 and T47D). Using our technique, we were able to pinpoint the effects of HDAC on a specific histone mark. RNA-seq identified genes upregulated in Entinostat were involved mostly in exocytosis and axon guidance pathways. Comparison of genomic distribution of ChIP-seq peaks between DMSO control and Entinostat-treated MDA-MB-231 breast cancer cells revealed a modest increase of binding sites at intergenic regions and promoter regions. Although most H2A.Zac sites were at promoters, differential binding analysis identified that more than 80% of binding sites were upregulated at intergenic regions and that 60% of the down-regulated binding sites were at the promoters in Entinostat-treated MDA-MB-231 cells compared to control. Moreover, integrative analysis of RNA-seq and ChIP-seq data suggested that histone H2A.Zac’s role in regulating gene expression through positive regulation of gene regulation, axon guidance pathways and specific genes involved in tumor suppressor pathways. Our findings suggest that H2A.Zac might reprogram epigenetics by re-expressing tumor suppressor genes and thereby inhibiting cancer progression.
Committee Chair
Benjamin Garcia
Committee Members
Elizabeth Draganova; Gabor Egervari; Michael Major; Michael Meers
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Biochemistry)
Document Type
Dissertation
Date of Award
4-17-2026
Language
English (en)
DOI
https://doi.org/10.7936/588v-n913
Recommended Citation
Karki, Rashmi, "Mass Spectrometry Profiling and High-Throughput Sequencing of HDACi-Induced Histone Modification Changes" (2026). Arts & Sciences Graduate Student Theses and Dissertations. 3782.
The definitive version is available at https://doi.org/10.7936/588v-n913